The molecular mechanism for morphine tolerance has not been firmly established yet. The current model of [j-opioid receptor (MOR) desensitization via the &-Arrestin pathway cannot account for the numerous observations that other neurotransmitter receptor activities, such as NMDA, could contribute to morphine tolerance. The activity of other opioid receptors, such as the 6-opioid receptor (DOR), could be implicated in morphine tolerance development also. Since morphine can activate and desensitize DOR during prolonged treatment, our working hypothesis is that the post-signaling events occurring within the DOR-containing neurons during morphine treatment contribute to tolerance development. Our working hypothesis also is that morphine has post-signaling events distinct from those of other opioid agonists. In order to demonstrate these hypotheses, agonist-dependent signaling events will be established for morphine activation of DOR. In our studies with MOR signaling, we have demonstrated that morphine differs from other agonists in its pathway to activate ERK1/2. Agonists such as etorphine activate ERK1/2 via the B-Arrestin-dependent pathway, while morphine activates ERK1/2 via the PKC-dependent pathway. This divergent activation results in differential translocation of the activated ERK1/2 and the transcripts produced. Therefore, the signaling pathway and the post-signaling events of morphine in cell models expressing DOR will be established. The possible involvement of the PKC-dependent pathway on morphine-mediated DOR activation of ERK1/2 will be studied. The specific PKC subtypes involved will be defined. The reasons for the differences among agonists in selecting a pathway will be investigated by monitoring protein-protein interactions using a novel protease assay system. Parallel studies will be conducted with primary neuronal cultures. The blockade of specific PKC subtypes in DOR-expressing neurons on in vivo morphine tolerance development will be explored. By selectively inactivating the morphine signaling pathway, and subsequently its post-signaling events in DOR-containing neurons, possible blockade of morphine tolerance without altering morphine activities in MOR containing neuron could be accomplished.